Collagen type I (rat tail tendon) was obtained from Beckton Dickenson (Oxford, UK) and Matrigel from Stratech Scientific (Luton, UK). Dulbecco's Eagle's Medium (DMEM), Ham's F-12 and fetal calf serum (FCS) were from Gibco (Life Technologies, Paisley, UK). Human Activin AA was a kind gift from the National Hormone and Pituitary Program, NIDDK (Dr Parlow). Rabbit anti-mouse streptavidin-FITC and biotinylated rabbit anti-mouse-Ig were from Dako (High Wycombe, UK). Human recombinant IGF-I was obtained from Genentech (San Francisco, USA), recombinant human TGFβ1 from R&D Systems (Abingdon, UK). Monoclonal anti-cytokeratin 7 was from Dako (Ely, UK). Monoclonal antibody αIR3 to IGFR1 was from Calbiochem (Nottingham, UK). Monoclonal Ab-1 to IGFR1 was a kind gift from Prof K Siddall, University of Cambridge, UK. Monoclonal antibody 15C9 to IGF-I was as described previously .
Normal first trimester (8–12 week) placenta was obtained by elective surgical or medical termination of pregnancy. Tissue was collected in PBS supplemented with antibiotic and antimycotic solution (AAM; Sigma, Poole, UK) prior to dissection. The results obtained using the explant system were independent of collection method.
The explant culture method was as described in Aplin et al. Dissected mesenchymal villous tissue was arranged radially on 80 μl drops of collagen type I gel, covered with 20 μl of serum-free medium (SFM; an equal mixture of DMEM and Ham's F12 (Gibco, Paisley, UK), supplemented with 1% AAM) and incubated overnight at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 1 ml SFM and incubated at 37°C, 5% CO2.
After an initial period of 24 h during which cytotrophoblast proliferation occurs at the villous tips to produce anchoring cell columns , growth factors or fibroblast conditioned SFM were added to the culture medium. IGF-I (10 ng/ml), TGF-β1 (4 ng/ml), activin AA (10 ng/ml) or IGF-II (10 ng/ml) were added daily to the culture medium and subsequent growth characteristics monitored by light microscopy. In addition, explants were cultured with monoclonal antibody Ab-1 (1 μg/ml) to human IGF receptor 1 (IGFR1) added daily (with or without 10 μg/ml IGF-I), or in the presence of fibroblast conditioned SFM depleted of IGF-I using immunoaffinity chromatography (see below). Cultures were continued up to 7 days.
This assay has the significant advantage of allowing migratory behaviour to be observed in living trophoblast, because migration occurs largely across the surface of the gel. Thus whole mount examination can be undertaken without the need to produce thin sections. Outgrowths were examined qualitatively taking into account size, distance, the morphology of the constituent cells and their inter-relationships including cell-cell adhesion and sheet integrity. Semi-quantitative evaluation of cytotrophoblast outgrowths from explants was carried out by analysing individual sites at villous tips. Images of live cultures were captured using a an imaging system (KS400, Kontron Elektronik GmbH, Eching, Germany) coupled to a Leitz Diavert microscope. Outgrowth distance was evaluated on a scale of 0 (no outgrowth) to 3 (outgrowth > 1 mm from tissue edge). Cell morphology was described on a scale of 0 (tightly packed cells throughout the outgrowth) to 3 (majority elongated and radially-oriented cells). Note that the number of cells present is not evaluated, as this varies widely between sites and is critically dependent on the size of the area of contact between tissue and gel at the start of the explant, a parameter that varies with the shape of the villus. The two estimates were added together to produce a value in the range 1–6 for each site. At least 3 experimental series with replicates from different placentas were used for each treatment.
Fibroblasts were isolated and characterised from first trimester tissue as described previously  and routinely passaged in monolayer. In order to produce conditioned medium, cells just below confluence were incubated in serum-free medium for 3 days. IGF levels in this medium were measured using our previously reported RIA . Serum-free fibroblast conditioned medium was depleted of IGF-I by affinity chromatography. 1 ml of anti-IGF-I antibody 15C9 (1 mg/ml ) was coupled to activated Sephacryl, then the solid phase incubated with conditioned SFM for 16 h at 4°C. IGF-depleted medium was harvested by centrifugation at 2800 rpm,10 min, and the supernatant was filter-sterilised, aliquotted and stored at -20°C. RIA was used to confirm removal of IGF.
Fresh placental tissue and 4–7 day old explant cultures were snap frozen in OCT (Tissue-Tek, Cambridge, UK) and stored at -80°C. 6 μm cryosections were cut and mounted on poly-L-lysine-coated slides for immunocytochemical analysis. Cryosections were stained essentially as described previously . IGFR1 was localised with antibody αIR-3 (5 μg/ml). Antibody to cytokeratin 7 was used as a positive control marker to verify the identity of trophoblast .
IGF-I was measured by a previously reported modification of the functional separation method  in which excess IGF-II blocks the interference from IGFBPs . Briefly, samples were diluted 1:100 in acidic sample buffer and then incubated with 100 μg/l IGF-II, 125I-IGF-I (20 000 cpm) and anti-IGF-I antibody (1:4000 dilution of clone BPL-M23) at 4C, pH 7.8 for 16 h. Separation of bound and free radioligand was performed with anti-mouse IgG linked to cellulose (Sac-Cel; IDS, Tyne & Wear, UK) for 1 h at 37C, followed by centrifugation at 1000 g for 10 minutes at 4C. The detection limit, defined as 10% displacement of the binding at zero dose of IGF-I was 0.08 ng/tube and the inter- and intra-assay coefficients of variation were 5.2–7.4% and 4.0–5.7% respectively. Cross reactivity with IGF-II and insulin was <2% for both peptides.